The long term goal of this proposal is the development of tools enabling high throughput single-molecule fluorescence spectroscopy. The specific aims of this proposal are: (1) Develop an optical setup for 3 color, 48 spot excitation and detection. (2) Develop a 48 pixel custom single-photon avalanche photodiode array (48p-SPADA). (3) Develop field-programmable-gate-array (FPGA) data reduction algorithms for up to 144 channels single-molecule burst analysis. (4) Develop high-throughput fluorescence (cross)-correlation spectroscopy data analysis algorithms in graphical processing unit (GPU). (5) Study fast and transient events in transcription initiation and elongation, focusing on the conformation and interactions of 70 with the bacterial RNAP holoenzyme. The expected outcome of this proposal is an increase in throughput for single molecule fluorescence techniques of a factor 48. This will include most single-molecule fluorescence techniques such as single-molecule fluorescence resonant energy transfer (smFRET) and microsecond alternating laser excitation (s-ALEX) as well as fluorescence correlation spectroscopy (FCS). The impact of these developments will be twofold. First, single-molecule fluorescence spectroscopy methods will become attractive to a larger research community, which will be able to use is as an efficient discovery and analytical tool. Additionally, it will give access to new temporal regimes that are unattainable with the current technology. Second, the leap in throughput (by a factor 48) will render this technology attractive for point-of-care diagnostics and large scale screening assays used by the biotechnology and pharmaceutical industry. For these reasons, this proposal is very likely to significantly impact the progresses of many areas of biological research including drug discovery and development as well as diagnostics, thus helping increase our understanding of life processes and lays the foundation for advances in disease diagnosis, treatment, and prevention.